The isolation of the gene coding for the regulatory light chain of smooth muscle offers an opportunity to examine the controls over the expression of the protein whose function is important for both smooth muscle cell and non-muscle cells. Comparing this gene's regulation of the thick filament regulatory phenotype with those genes of the thin filament regulatory system of cardiac and skeletal muscles will help elucidate the basic mechanisms controlling these two differentiated contractile systems. Working on this project Dr. Grant will continue his training in molecular and cellular biology under the sponsorship of Dr. Bernardo Nadal-Ginard who has used these approaches to the study of the skeletal and cardiac contractile systems. The gene for the 20,000 dalton regulatory light chain of smooth muscle will be isolated by screening a rat genomic library using the full length regulatory light chain cDNA already isolated by the applicant. The structural organization of the gene will be defined by a combination of sequencing and restriction mapping. A variety of tissues including smooth muscle, non-muscles and straited muscles will be examined by northern blots and S1 mapping analysis to study the tissue specific expression of this gene. In this manner the identification of either a gene family or the use of alternative splicing within only one or two genes can be made. The elucidation of the basic genetic organization will enable the applicant to identify the cis-acting control regions of the regulatory light chain gene. This will be done by constructing expression vectors using variable regions of the 5' flanking region of the gene combined with DNA-mediated cell transfection assays in a variety of cell types to define the minimal cis sequences required for tissue-specific transcription of the gene. Once identified and isolated these sequences will be compared to the corresponding sequences of the other contractile proteins in order to begin exploring the mechanisms controlling the expression of the contractile phenotypes in various cell and tissue type. The data generated during this phase of the research will provide the basis for identifying and characterizing trans-acting factors that are important in the generation of smooth muscle cell differentiation and gene-specific expression. The results obtained from this work should improve our understanding of smooth muscle cell bioloby and its alteration in a wide variety of disease processes.